Apoptosis is a cell death brought about positively by the cell per se under physiological conditions. Its morphological features are the chromatin condensation in the cell nucleus, fragmentation of the nucleus, the elimination of the cell cortical microvilli, and the condensation and fragmentation of cytoplasm. Cell cortical receptor Fas antigen triggering apoptosis, protease (caspase) indispensable for triggering apoptosis, and oncogene bcl-2 gene suppressing apoptosis, etc. have recently been demonstrated.
Oncogene bcl-2 is identified as such being activated by t(14;18) (q21;q32) chromosome translocations associated with follicular lymphoma, and is a unique oncogene displaying apoptosis-suppressing function. Various proteins that are homologous to Bcl-2 are called Bcl-2 family. Among Bcl-2 family, the presence of members with apoptosis-suppressing function represented by Bcl-2 and Bcl-xL, and members with apoptosis-promoting (or inducing) function represented by Bax and Bak are known.
The followings are also known: that the mitochondria plays a crucial role in apoptotic signal transduction, and the apoptotic changes in the mitochondria are regulated by Bcl-2 family proteins; that the mitochondrial membrane potential (ΔΨ) loss and cytochrome c release are inhibited by Bcl-2 and Bcl-xL both having apoptosis-suppressing functions (Proc. Natl. Acad. Sci. USA 95, 14681-14686, 1998. J. Exp. Med. 183, 1533-1544, 1996. Cell 91 , 627-637, 1997. Proc. Natl. Acad. Sci. USA 95, 1455-1459); and that the mitochondrial membrane potential (ΔΨ) loss and cytochrome c release are induced by Bax and Bak both having apoptosis-promoting functions (Proc. Natl. Acad. Sci. USA 95, 4997-5002, 1998. J. Cell Biol. 143, 217-224, 1998. Proc. Natl. Acad. Sci. USA 95, 14681-14686, 1998).
Further known are that Bax- and Bak-dependent changes of membrane permeability are mediated by poly-protein channel called the permeability transition (referred to as PT hereinafter) pore (Proc. Natl. Acad. Sci. USA 95, 4997-5002, 1998. Proc. Natl. Acad. Sci. USA 95, 14681-14686, 1998. Science 281, 2027-2031, 1998), and that the poly-protein channel is thought to consist of VDAC (voltage-dependent anion channel) which is also called mitochondrial porin, the adenine nucleotide translocator (ANT), cyclophilin D, and some other molecules (J. Bioenerg. Biomembr. 26, 509-517, 1994. Biochem. Biophys. Acta 1241, 139-176, 1995).
VDAC is a small protein abundantly existing on the outer mitochondrial membrane, which was found to form a large (2.6 nm) voltage-dependent pore when VDAC-liposomes were formed on being incorporated by planar lipid bilayers (J. Membr. Biol. 111, 103-111, 1989). VDAC is thought to physiologically function as the pathway for various substances including ions and intermediate metabolites to get into and get from the mitochondria. Further, the present inventors have recently demonstrated that Bax and Bak interact with the PT pore (Proc. Natl. Acad. Sci. USA 95, 14681-14686, 1998. Science 281, 2027-2031, 1998). It has also been reported that Bax interacts directly with ANT (the adenine nucleotide translocator) and sensitizes ANT and PT pore-liposomes to atractyloside, i.e. an ANT ligand (Science 281, 2027-2031, 1998).
Bcl-2 and Bax are highly involved in oncogenesis. In follicular lymphoma or diffuse large cell lymphoma with t(14; 18) translocations, and in chronic lymphatic leukemia either with t(18;22)(q21;q11) or t(2;18)(q11;q21) translocations, Bcl-2 expression is deregulated by respective translocations to immunogloblin heavy chain and light chain regions, and Bcl-2 promotes suppression of cell death, contributing to oncogenesis. Meanwhile, Bax functions as a tumor-suppressor gene, suggesting its involvement in human tumor, as the mutation of Bax genes (BH1 and BH3 regions) are observed with high frequency in the tumor cell strain of blood cell system.
Bcl-2 is also deeply concerned with neurodegenerative diseases. SMN (survival motor neuron) protein is a causal gene product of spinal muscular atrophy; a genetic disease accompanied with degeneration of motor nerves and crisis of the disease is deeply associated with the decrease in Bcl-2 function due to SMN-deficiency.
The subject of the present invention is to provide the screening method for apoptosis-suppressing substance or apoptosis-promoting substance, whose future applications to pharmaceuticals or diagnostic drugs are expected since Bcl-2 family having apoptosis-suppressing or -promoting activities are deeply involved in various diseases, and to provide apoptosis-suppressing substance or apoptosis-promoting substance.